Long-lasting potentiation at hippocampal synapses is a form of synaptic plasticity that is thought to be a likely substrate of mammalian learning and memory. To investigate mechanisms of the potentiation, we recently examined immunoreactivity for pre- and postsynaptic proteins during potentiation in dissociated cultures ofhippocampal neurons (Antonova et al., 2001). Consistent with recent studies, we found that there is an increase in clusters (puncta) of postsynaptic proteins (GluR1, PSD95) at the onset of long-lasting potentiation. However, we also found that these postsynaptic changes are accompanied by a rapid increase in clusters of presynaptic proteins (synaptophysin, synapsin I, synuclein) and sites where the pre- and postsynaptic proteins colocalize and therefore might participate in functional synapses. We now propose to extend these findings in two ways. First, we will examine the significance of the new presynaptic puncta by testing whether they participate in new functional synapses immediately, or whether they are part of structures that mature into functional synapses with the passage of time. Because the assembly of colocalized pre- and postsynaptic puncta is reminiscent of early synaptogenesis and the later stages of potentiation are accompanied by the growth of new synapses, it is attractive to think that the new puncta might represent a step in that process. We therefore propose to investigate whether long-lasting potentiation involves the coordinated assembly of a variety of synaptic components, as occurs during synaptic development. Second, we will begin to investigate retrograde messengers that may be involved in the formation of the new puncta, including freely diffusible molecules such as NO, extracellular messengers such as neurotrophins, and adhesion molecules. We will also investigate possible presynaptic effectors of these messengers, focusing on ones that are known to be involved in regulating the actin cytoskeleton, which plays a critical role in the increase in presynaptic puncta. These studies should provide new information about the functional significance and molecular mechanisms of a novel aspect of synaptic plasticity that may be important for learning and memory.